There is increasing evidence of the importance of MC - T cell crosstalk in regulating the immune response. MC effector activity is suppressed by Tregs (Gri et al., 2008
) and Tregs can also use MCs as mediators of immune suppression (Lu et al., 2006
). MCs have also been implicated in promoting CD4+
T cell expansion (Piconese et al., 2009
; Stelekati et al., 2009
). Herein, we describe a regulatory mechanism in which secretion of IL-2 by MCs suppresses chronic allergic dermatitis by enhancing the proportion of Tregs at the site of inflammation and decreasing the fraction of activated T cells at this site.
It has been reported that MCs can either promote (Biedermann et al., 2000
; Bryce et al., 2005
; Dudeck et al., 2011
; Suto et al., 2006
) or dampen contact hypersensitivity (Depinay et al., 2006
; Grimbaldeston et al., 2007
; Hart et al., 1998
), the latter being mediated primarily through MC production of IL-10. Dudeck and coworkers recently reported (Dudeck et al., 2011
) that MCs are key promoters of contact hypersensitivity and not suppressors of this response. They induced contact hypersensitivity in a manner similar to that reported in the Grimbaldeston study (Grimbaldeston et al., 2007
), using a mouse model (Mcpt5-Cre+iDTR+
) in which connective tissue type MCs are selectively depleted. Their findings showed that the absence of connective tissue type MCs impaired migration of DCs to the lymph nodes and caused a marked reduction in contact hypersensitivity. They proposed that previous studies in mast cell deficient mice (KitW-sh/W-sh
) showing MC protective effects were flawed due to potential immunological abnormalities in these mice. While it is clear that KitW-sh/W-sh
mice have various abnormalities (Grimbaldeston et al., 2005
; Wolters et al., 2005
), such overt dismissal for the role of MCs as suppressors should be cautioned by the fact that the particular disease model, mouse strain or genetics, route of sensitization and challenge, and strength of stimulus can all influence the immune response. In agreement with the Dudeck study, we (data not shown) and others (Norman et al., 2008
; Suto et al., 2006
) have seen that MCs can promote acute contact hypersensitivity in KitW-sh/W-sh
mice in models where strength of the stimulus may be modest. Interestingly, Norman and colleagues (Norman et al., 2008
) showed that induction of contact hypersensitivity by a weak versus strong stimulus was a determinant of whether MCs induce or suppress disease, respectively. In our study, we utilize a model that differs from that used by Grimabaldeston and Dudeck in that repeated challenges of oxazolone on the ear were done for a period of approximately 28 days. This chronic model shows many of the characteristics of human AD (Leung et al., 2004
) including the presence of both Th1 and Th2 cell cytokines. Our finding of MC-mediated suppression is consistent with the results of Norman et al. (Norman et al., 2008
) where a strong stimulus induced both Th1 and Th2 cell cytokines and resulted in a protective role for MCs. In addition, our findings also show that MCs can act to cause suppression from tissues (spleen) distal to the site of inflammation. Thus one cannot exclude that the presence of mucosal MCs, which are seemingly not depleted in the diptheria toxin-induced Mcpt5-Cre+iDTR+
mice, could also influence observed responses.
Our deduction that the spleen represents a site involved in MC regulatory function is based on several findings. Splenectomized mice had a diminished inflammatory response and anti-CD25 treatment of these mice failed to exacerbate disease. This demonstrated the contribution of the spleen in oxazolone-induced inflammation and showed that the effect of anti-CD25 was dependent on the spleen. The effect of anti-CD25 treatment was independent of MCs since oxazolone-induced dermatitis was markedly exacerbated in the MC-deficient KitW-sh/W-sh
mice. Intravenous MC reconstitution of KitW-sh/W-sh
mice, which causes engraftment of the spleen, restored regulation of disease despite no engraftment of MCs in ear pinna. It is important to note that i.v.
reconstitution of KitW-sh/W-sh
mice with MCs engrafts several other sites (Grimbaldeston et al., 2005
; Wolters et al., 2005
), including LNs (a key site of MC - T cell interactions (Gri et al., 2008
; Piconese et al., 2009
)), and thus these sites may be contributory. Migration of MCs to the spleen was dependent on IgE accumulation in the spleen, and mirrored both the increased production of IL-2 by MCs in the spleen and suppression of disease. The precise mechanism underlying the increase of MCs in the spleen has yet to be elucidated. It does not appear to involve FcεRI, as mice deficient in FcεRIβ (and thus unable to express FcεRI) showed increased numbers of MCs in the spleen following oxaxzolone challenge (data not shown). However, antibody blockade of CD23 inhibited the increase of splenic MCs (data not shown), suggesting the possible involvement of this low affinity IgE receptor. IgE engagement of CD23 on monocytes (which infiltrate the spleen upon oxazolone challenge (Sugiura et al., 2010
)) has been shown to induce secretion of CCL3 (Ezeamuzie et al., 2009
), a potent chemoattractant for MCs. Collectively, these findings demonstrate a connection between the spleen and the skin in modulating chronic allergic dermatitis, and the putative trafficking of Tregs between the two sites warrants further investigation. It should be noted that the spleen has also been shown to be essential for the induction of antigen-specific allergic diarrhea due to mobilization of primed splenic CD4+
T cells to the large intestine (Kurashima et al., 2007
). Moreover, migration of MCs to the spleen is a well described phenomena, as acute models of DNFB (Wang et al., 1998
) and peptidoglycan (Matsui and Nishikawa, 2006
) dermatitis, and a model of helminth infection (Friend et al., 2000
) showed increased numbers of MCs in the spleen. MC-mediated regulation of disease severity at a location distal to the site of inflammation was also shown in a mouse model of experimental autoimmune encephalomyelitis, where MCs exacerbated disease following adoptive transfer without repopulating the inflammatory site, i.e. the central nervous system (Tanzola et al., 2003
). Thus, we propose that MC migration to the spleen may be important beyond allergic skin disease.
Localization of Treg - MC interactions to a site such as the spleen may provide an explanation to the observation that exogenous administration of IL-2 in post-sensitized mice did not modify disease course, whereas MC-derived IL-2 alleviates chronic disease. We hypothesize that a systemic bolus of IL-2, while disease is ongoing, may stimulate both regulatory and effector T cells that express the IL-2R thus failing to alter the course or severity of disease. Conversely, a systemic bolus prior to disease would target those cells constitutively expressing the IL-2R, which are primarily Tregs (Malek and Bayer, 2004
), and cause amplification of their activity thus suppressing the disease. The production of IL-2 by MCs at a distal lymphoid organ may serve a similar role in compartmentalizing the effect of IL-2 during the disease course. Our data do not define the numbers of MCs required in order to maintain normal regulation in dermatitis. In fact, comparable degrees of regulation were obtained in C57BL/6 as in KitW-sh/W-sh
mice following i.d.
injection of the latter with MCs. Although in C57BL/6 mice MC numbers in the spleen (per 20mm2
) were in the range of 5-10, i.d.
showed four to five fold greater numbers and for i.v.
several hundreds (Grimbaldeston et al., 2005
)(and data not shown). Thus, the absolute numbers of MCs migrating to the spleen does not appear to affect the onset or extent of the suppressive regulatory effect on disease. We also theorize that in the early phase of dermatitis (or throughout disease course in the case of KitW-sh/W-sh
mice) IL-2 is provided by cells other than MCs. This is supported by the finding of a small population of splenic CD4+
T cells (approximately 0.8%) that stain positive for IL-2 in naïve WT mice (data not shown). However, unlike MCs, this population of T cells did not expand during disease course. In addition, we detected IL-2 producing cells in the spleens of non MC-reconstituted KitW-sh/W-sh
mice with dermatitis. We cannot formally exclude that some of the observed responses in KitW-sh/W-sh
mice might result from other described abnormalities in these mice beyond the MC compartment (Wolters et al., 2005
). Nonetheless, one must conclude from the MC reconstitution of KitW-sh/W-sh
mice that IL-2 expression in MCs is vital for late stage disease suppression, but it is not the rate-limiting factor in initiating regulatory control.
Whereas our results indicate that Tregs and MCs exert comparable effects in suppressing the response to repeated oxazolone challenge, the overall impact on immune homeostasis differs greatly. This is exemplified by comparing the severe phenotype resulting from Treg deficiency in Foxp3sf
mice (Brunkow et al., 2001
) and the mild phenotype associated with the absence of MCs in KitW-sh/W-sh
mice (Grimbaldeston et al., 2005
). Whereas Foxp3sf
mice have a life span of 16-25 days, KitW-sh/W-sh
have a nearly normal life span. Foxp3sf
mice can develop a spontaneous and severe eczematous skin disease (which made them unsuitable for our studies) whereas KitW-sh/W-sh
mice may show mild skin lesions in their late life. In fact, the data argue that Tregs do not require MCs to suppress disease at early stages (prior to the fourth or fifth challenge) rather their role appears to be in providing help to Tregs once disease has reached almost maximal severity. This view is supported by the findings that inactivation of Tregs increases the numbers of splenic MCs in the spleen and that consequent IL-2 production supports Treg numbers at the site of inflammation.
In summary, we propose that MC - Treg cell communication during the course of oxazolone-induced chronic allergic dermatitis is vital to disease regulation. Our data argues that the spleen plays a role in the inflammatory process and serves as a site where MCs are recruited as second line regulators in an IgE-dependent manner. By producing IL-2, MCs serve to maintain the Treg:Teff cell ratio at the site of inflammation, which is manifested by control of disease severity. Thus, the findings show that MCs play an overall protective role in late stages of chronic allergic dermatitis and suggest that failure of MC function in this disease leads to increased severity.